Project Summary ! Ca2+-activated Cl- channels (CaCCs) open in response to increases of intracellular Ca2+ and selectively conduct Cl- and other anions. To date, two families of CaCCs, namely BESTROPHINs and TMEM16s, have been identified, among which three members- BESTROPHIN1 (BEST1), TMEM16A and TMEM16B have been proposed to function in human retinal pigment epithelium (RPE). BEST1 is predominantly expressed in RPE and genetically linked to a spectrum of retinal degenerative disorders. However, Best1 knockout mice did not display any retinal phenotype or Cl- current abnormality, arguing against the idea that BEST1 is an essential CaCC in RPE. Although human and mice may have fundamental differences on the genetic requirement of CaCCs in their RPEs, no direct evidence has been documented to support this hypothesis. TMEM16A and TMEM16B, on the other hand, are widely expressed in a variety of cell types including RPE. Their CaCC roles in RPE were suggested by studies in animal models and cell lines, but have not yet been examined in human RPE. Therefore, the physiological contributions of the three candidate CaCCs in human RPE still remain a mystery. This deficit is mainly due to the technical challenges: 1) the accessibility to native human RPE cells is very limited, and it is hard to perform gene manipulation with them; 2) currently available Cl- channel inhibitors cannot effectively distinguish BEST1, TMEM16A and TMEM16B. In this proposal, we aim to use multidisciplinary approaches, including CRISPR/Cas9-mediated genome editing, stem cell technology, whole-cell patch clamp and X-ray crystallography, to define the functional CaCC(s) in human RPE (in Aim 1), and to conduct an unprecedented mechanistic investigation on the CaCC activity and physiological role of BEST1 (in Aims 2 and 3). Our proposed work will reveal basic principles of CaCC function and regulation in human RPE, thereby making significant contributions to multiple fields of research including calcium signaling, ion transport, membrane protein structure and retinal physiology. Moreover, the pipelines established in this work can be generally applied to study ion channels and/or genes of interest in other human organs.